Novel Use of Von Willebrand Factor

ABSTRACT

The present invention relates to a von Willebrand factor composition for the prevention and/or the treatment of haemorrhage and/or bleeding in patients with mechanical circulatory support.

The present invention relates to a novel use of von Willebrand factor,and in particular to the treatment and/or the prevention of haemorrhageand/or bleeding in patients with mechanical circulatory support.

Mechanical circulatory support is an intracorporeal or extracorporealdevice capable of replacing, either totally or partially, thecirculatory functions normally performed by the heart. Its objective isto maintain or restore the main functions of the organism, and inparticular to ensure, as the case may be, a uni- or biventriculardischarge or to completely take over the function of the failing heart.This type of device is prescribed when the patient's haemodynamic statusrequires it, in particular in three specific indications:

-   -   in patients waiting for a heart transplant in order to avoid a        deterioration in the haemodynamic status of the patient due to a        prolonged wait on the transplant list;    -   in patients awaiting recovery, in particular in patients having        suffered viral myocarditis, a drug overdose or having a low        cardiac output after heart surgery under extracorporeal        circulation;    -   in patients who would possibly be candidates for a heart        transplant but who unfortunately present a contraindication for        such a surgical intervention (pulmonary hypertension, neoplasia        in remission but not cured, age).

The first circulatory support systems were all of the pneumatic type.They were all composed of a rigid outer shell inside which there iseither a bag or a diaphragm made of polyurethane. Mechanical orbiological valves ensured a unidirectional flow. They were connected toa drive console which was generally very large and cumbersome,restricting the movements the patient could make. Compressed air camefrom a wall socket at the hospital (CardioWest®) with the possibility ofoperating temporarily with bottles of compressed air situated inside theconsole. All of the other systems were connected to consoles insidewhich air was compressed by a compressor. The operating principle ofthese devices is simple: a low pressure around the bag or under thediaphragm ensures that the prosthesis is filled. In reverse, during theejection phase, compressed air compresses the bag or pushes back thediaphragm, thus making possible the rhythmic ejection of the blood. Allof these ventricles therefore provide a pulsed flow. Among thesepneumatic ventricles there are those that are paracorporeal which can beused for univentricular support (most often on the left) orbiventricular support, and implanted ventricles (Total Artificial Heartfrom CardioWest®). All of these ventricles provide an excellenthaemodynamic performance and can be used over long periods making itpossible to wait for a transplant under the best possible conditions.The system used most often is, without doubt, the Thoratec® system whichhas a novel drive console (TLC-II) which improves the patient's comfortand also allows the patient to leave hospital and to return home.

Ventricles of the last generation operate with electrical energy. In the1980s and 1990s there were two electro-mechanical ventricles implantedbetween the top of the left ventricle and the ascending thoracic aorta(Novacor®, HeartMate XVE®). The pump was implanted behind the muscles ofthe abdominal wall and an electric cable connected this pump to acontroller and to a power source. The flow delivered was of the pulsedtype. The Novacor® is no longer available and the HeartMate® is hardlyused any more, at least in Europe.

The last generation of implantable pumps is of the rotary type. Theseimplantable pumps can be classified in two groups:

-   -   centrifugal pumps such as DuraHeart® or HeartWare® and,    -   axial pumps such as HeartMate II®, MicroMed DeBakey®,        BerlinHeart Incor® or Jarvik 2000®.

These pumps delivering a continuous, not a pulsed, flow do not needvalves to ensure a unidirectional flow. In the case of most of theserotary pumps, the rotor turns about a shaft.

However, it has been found that patients with this type of implantablepump, delivering a continuous, not a pulsed, flow develop an acquiredvon Willebrand disease. In fact, in these patients, a significant fallin the fraction of high-molecular weight multimers of von Willebrandfactor has been found, thus causing mainly gastro-intestinal bleeding,which doctors have difficulty treating.

Fisher et al. (Transfusion, 2015; vol. 55(1), pp. 51-54.) reports thecase of a patient with mechanical circulatory support using a continuousflow pump (HeartMate II), repeatedly suffering severe gastro-intestinalbleeding following the implantation of the pump. After the failure ofconventional treatments, this patient received 80 IU/kg of body weightof von Willebrand factor on day 17, 18 and 19 after the introduction ofthe mechanical circulatory support. The treatment with von Willebrandfactor was maintained and a new dose of 80 IU/kg of body weight wasagain administered to the patient on days 21, 23, 27 and 30 after theoperation. However, on day 41, the doctors found that the patient'sileum appeared hyperdense, suggesting active haemorrhage in this region.A new administration of von Willebrand factor was then carried out everytwo days from day 41 to day 53 after the operation. On day 57 after theoperation, the doctors discovered three angiodysplasic lesions in theduodenum, lesions likely to cause serious haemorrhage. As a result,therefore, despite a massive administration of von Willebrand factor ondays 17 to 19 after the operation, the doctors could not stop theoccurrence of a new gastro-intestinal (ileum) haemorrhage.

Cushing et al. (Transfusion, 2012; vol. 52, pp. 1535-1541) report thecase of a patient with mechanical circulatory support using a continuousflow pump (HeartMate II) while waiting for a heart transplant. Thepatient developed a haemorrhage in the duodenal bulb 9 days after theintroduction of the mechanical circulatory support device. Thishaemorrhage was treated with a blood transfusion and the administrationof aminocaproic acid. 47 days after the intervention, the patient hadseepage in the gastric area and in the duodenal bulb as well as activebleeding at the duodenojejunal junction. On the 49^(th) day after theplacement of the mechanical circulatory support device, the patientreceived a dose of 60 IU/kg of body weight of a pharmaceuticalcomposition comprising von Willebrand factor and factor VIII every eighthours, in three doses while continuing the administration ofaminocaproic acid. Treatment with the composition of von Willebrandfactor and factor VIII was continued for 33 days with decreasing doses.However, the patient developed bruises around the left eye and athrombus at the pump a few days later, resulting in cessation of thetreatment. The authors concluded that the treatment with the compositionof von Willebrand factor and factor VIII made it possible to treatgastro-intestinal haemorrhage and/or bleeding, but that this treatmentrequires very close monitoring of thromboses in the pump circuit, inparticular because of the presence of factor VIII in the therapeuticconcentrate. As a result, therefore, although it was possible to treathaemorrhages in the patient being studied with an administration of thecomposition of von Willebrand factor and factor VIII, the administrationof this product can lead to the occurrence of thrombosis.

Consequently, it would appear necessary to provide a method for theprevention and/or a method for the treatment of haemorrhage and/orbleeding which could be applied to patients with mechanical circulatorysupport, in order to prevent the occurrence of these haemorrhages and/orof this bleeding, without the fear of causing an additional thrombosisand without the fear of a possible relapse, i.e. without the fear ofdeveloping new haemorrhages or new bleeding.

The inventors have shown, surprisingly, that the administration of apharmaceutical composition comprising von Willebrand factor at a fixeddose and under specific conditions made it possible to prevent the risksassociated with the introduction of mechanical circulatory support. Moreparticularly, the inventors have shown that this preventativeadministration of von Willebrand factor made it possible, surprisingly,to reduce by more than 60% the risks associated with the introduction ofmechanical circulatory support, namely to reduce by more than 60% theoccurrence of haemorrhage and/or bleeding in these patients. Thispreventative administration of von Willebrand factor preferably makes itpossible to reduce the occurrence of haemorrhage and/or bleeding inthese patients by more than 70%, preferably 80%, preferably 90%.Advantageously, this administration of von Willebrand factor makes itpossible to permanently eliminate any risk of haemorrhage and/orbleeding in patients with mechanical circulatory support, including therisks of relapse.

In another advantageous embodiment, the administration of apharmaceutical composition comprising von Willebrand factor according tothe invention makes it possible to reduce the frequency of clinicallysignificant bleeding in comparison with usual care.

The invention therefore relates to a pharmaceutical compositioncomprising von Willebrand factor intended to be used in the preventionof haemorrhage and/or bleeding in patients with mechanical circulatorysupport.

The term “bleeding” means an (internal or external) blood flow outsidethe natural blood circulation of low quantity. When the loss of blood issignificant and likely to be life-threatening it is then called“haemorrhage”. The bleeding or haemorrhage may be through the skin,through a natural orifice or may be internal. The term “clinicallysignificant bleeding” means an internal or external bleeding leading todeath or to a prolonged period in hospital, or requiringre-hospitalization, surgery or the transfusion of at least 3 units ofred blood cells, or causing an increase in haemoglobin to a level higherthan 3 g/dl, or which is resistant to conventional treatment methods.

Within the meaning of the present invention, by “pharmaceuticalcomposition” is meant a pharmaceutical composition in liquid form, inparticular in the form of a solution or a suspension as well as apharmaceutical composition in powder form. This pharmaceuticalcomposition comprises at least one active ingredient, the von Willebrandfactor and pharmaceutically acceptable excipients.

The term “von Willebrand factor” or “vWF” includes the polypeptidescomprising the sequence of wild-type human von Willebrand factor or ofvon Willebrand factor derived from another species (for example bovine,porcine, canine or murine). It also comprises the natural allelicvariations of von Willebrand factor which can exist and any form ordegree of glycosylation or other post-translational modification. Theterm “von Willebrand factor” also includes the variants of vonWillebrand factor which have the same or a higher biological activitywith respect to the activity of the wild form, these variants inparticular have at least 70%, preferably 75%, preferably 80%, preferably85%, preferably 90%, preferably 92%, preferably 94%, preferably 95%,preferably 96%, preferably 97%, preferably 98%, preferably 99%,preferably 100% homology with the nucleotide sequence of wild-type vonWillebrand factor. Advantageously, the von Willebrand factor is a humanvon Willebrand factor.

In an advantageous embodiment, the pharmaceutical composition comprisingvon Willebrand factor intended to be used in the prevention ofhaemorrhage and/or bleeding in patients with mechanical circulatorysupport is depleted of factor VIII. The term “depleted of factor VIII”means that the composition of the invention does not contain factor VIIIor that it is present in a negligible quantity. In fact, the inventorsfound that the elimination of this clotting factor made it possible toreduce the risks of the occurrence of thrombosis, unlike the combinedFVIII/vWF pharmaceutical compositions already marketed such as, inparticular, the product constituted by 160 IU/ml von Willebrand factorand 66.6 IU/ml factor VIII.

Advantageously, the residual amount of factor VIII in the pharmaceuticalcomposition comprising von Willebrand factor according to the inventionis less than or equal to 10 IU/100 IU VWF:RCo. This level is measuredusing the von Willebrand ristocetin cofactor assay method (VWF:RCo) withrespect to the international standard of concentrated von Willebrandfactor defined by the World Health Organisation (WHO). The activity ofthe von Willebrand factor in the composition of the invention ispreferably 100 IU for 1 ml of reconstituted solution. The content ofresidual factor VIII in the composition according to the invention istherefore divided by a minimum of a factor of 4 with respect to theproducts on the market, thus making it possible to limit the risk ofthrombosis by controlling and reducing as far as possible the exogenoussupply of FVIII. The residual amount of factor VIII in thepharmaceutical composition comprising von Willebrand factor according tothe invention is preferably less than or equal to 10 IU/100 IU VWF:RCo,preferably less than 8 IU/100 IU VWF:RCo, preferably less than 6 IU/100IU VWF:RCo, preferably less than 4 IU/100 IU VWF:RCo. In a particularembodiment, the composition comprising von Willebrand factor is entirelyfree from blood clotting factor VIII.

In another embodiment, the pharmaceutical composition comprising vonWillebrand factor also comprises factor VIII in a non-negligiblequantity. Then, the factor VIII/von Willebrand factor ratio isadvantageously between 1/10 and 5/10 IU/ml.

In an advantageous embodiment, the pharmaceutical composition comprisingvon Willebrand factor intended to be used in the prevention ofhaemorrhage and/or bleeding in patients with mechanical circulatorysupport is depleted of ADAMTS13. The term “depleted of ADAMTS13” or“depleted of ADAMTS13 protein” means that the composition of theinvention does not contain any ADAMTS13 protein or that this ADAMTS13protein is present in a negligible quantity or that the ADAMTS13activity is negligible. In fact, the inventors found that this proteinhad a tendency to weaken the von Willebrand factor, and even to degradeit, and also that the activity of this protein is increased by theshearing forces induced by the mechanical circulatory support device.Thus, the absence of this protein or its presence in a negligiblequantity made it possible to reduce the risks of the occurrence ofhaemorrhage and/or bleeding in patients with mechanical circulatorysupport.

Advantageously, the pharmaceutical composition comprising von Willebrandfactor is depleted of ADAMTS13.

In another particular embodiment, the residual amount of ADAMTS13 in thepharmaceutical composition comprising von Willebrand factor according tothe invention is less than or equal to 0.15 μg/ml. Preferably, theresidual amount of ADAMTS13 is less than or equal to 0.10 μg/ml,preferably less than or equal to 0.05 μg/ml, preferably less than orequal to 0.01 μg/ml. The residual amount of ADAMTS13 is preferablybetween 0.01 μg/ml and 0.15 μg/ml.

Advantageously, the pharmaceutical composition comprising von Willebrandfactor does not contain ADAMTS13 activity.

In another particular embodiment, the residual level of ADAMTS13activity in the pharmaceutical composition comprising von Willebrandfactor according to the invention is less than or equal to the detectionlimit, or less than or equal to 0.03 ADAMTS13:Act [U/ml].

This level is measured using the method described in Kokame et al.(British Journal of Haematology, 2005, vol. 129, pp. 93-100), using FRET(fluorescence resonance energy transfer) and the fluorogenic substrateFRETS-VWF73 (Peptides International, Louisville, USA). The amount ofresidual ADAMTS13 in the composition according to the invention is atleast 2 to 3 times lower with respect to the products on the marketwhich comprise at least 0.13±0.07 ADAMTS13:Act [U/ml], thus making itpossible to avoid any risk of the occurrence of haemorrhage and/orbleeding in patients with mechanical circulatory support. In a preferredembodiment, the residual amount of ADAMTS13 in the pharmaceuticalcomposition comprising von Willebrand factor according to the inventionis less than 0.10 ADAMTS13:Act [U/ml], preferably less than 0.05ADAMTS13:Act [U/ml]. In a particular embodiment, the compositioncomprising von Willebrand factor is entirely free of ADAMTS13. Inanother embodiment of the invention, the quantity of ADAMTS13 in thecomposition comprising von Willebrand factor of the invention isadjusted in order to avoid the presence of ultra large multimers (>20mers). In this case, the ADAMTS13 can advantageously be of plasmaticorigin and copurified with the composition comprising von Willebrandfactor of the invention, or be of recombinant origin and advantageouslybe added to the composition comprising von Willebrand factor of theinvention of recombinant origin.

The distribution of the multimeric forms of von Willebrand factor isdefined after analysis of an electrophoresis gel making it possible toquantify the size of the mers (sub-units) in multiples of the monomersub-unit of 225 kD. Mers with a size of 225×2 to 225×15 are thusdescribed. By high-molecular weight multimers of the pharmaceuticalcomposition comprising von Willebrand factor is meant multimers startingfrom 10 monomers.

In a particular embodiment, the content of high-molecular weightmultimers in the pharmaceutical composition comprising von Willebrandfactor is close to that of plasma.

In another particular embodiment, the content of high-molecular weightmultimers in the pharmaceutical composition comprising von Willebrandfactor is sufficiently maintained in order to allow sufficient in vivoactivity of the pharmaceutical composition.

Advantageously, the pharmaceutical composition comprising von Willebrandfactor according to the invention has a content of high-molecular weightmultimers of at least 65%, preferably 70%, yet more preferably 75%,particularly preferably 80% of the total multimer content of vonWillebrand factor contained in the pharmaceutical composition. Thepresence of at least 65% of high-molecular weight multimers confers tothe composition according to the invention a better therapeuticefficacy.

The pharmaceutical composition according to the invention can comprisein addition one or more excipients, making it possible to stabilize thevon Willebrand factor and making it possible to solubilize thelyophilized forms of the von Willebrand factor.

In particular, the excipients can be chosen from:

-   -   a hydrophilic amino acid or one bearing a positively-charged        side chain,    -   optionally a hydrophobic amino acid,    -   a salt,    -   a protein stabilizer,    -   or a mixture thereof.

The hydrophilic (or polar) amino acids or the amino acids bearing apositively-charged side chain include lysine, arginine, histidine,glycine, serine, threonine, tyrosine, asparagine and glutamine. Amongthe hydrophilic amino acids or those bearing a positively-charged sidechain, arginine is preferably used, or one of the salts derivedtherefrom such as arginine hydrochloride or also arginine phosphate. Theamino acids such as glycine and/or lysine, or one of the salts derivedtherefrom such as lysine hydrochloride can advantageously be added. Inparticular, the hydrophobic amino acids include the following aminoacids: alanine, valine, leucine, isoleucine, phenylalanine, tryptophan,proline.

The salt can be an alkali metal salt, an alkaline earth metal salt or atransition metal salt. In particular, sodium citrate, calcium chlorideand zinc chloride can be mentioned by way of example as salt. In apreferred manner, the salt used is preferably sodium citrate or calciumchloride.

The protein stabilizer can be chosen from among the known proteinstabilizers, for example albumin and factor VIII. Advantageously, thepreferred protein stabilizer is albumin, preferably human albumin.

Advantageously, the composition comprises von Willebrand factor asactive ingredient and the following pharmaceutically acceptableexcipients: albumin, arginine hydrochloride, glycine, sodium citrate andcalcium chloride. More particularly, the composition can comprise:

-   -   human von Willebrand factor;    -   albumin, preferably human albumin;    -   arginine, optionally in the form of the hydrochloride;    -   glycine;    -   sodium citrate;    -   calcium chloride.

Reconstitution from powder in the form of an injectable preparation canbe carried out by adding water for injections (WFI water).

The term “prevention” or “prophylaxis” or “preventative treatment” or“prophylactic treatment” comprises a treatment leading to the preventionof a disease as well as a treatment reducing and/or delaying theincidence of a disease or the risk of it occurring. According to theinvention, the von Willebrand factor composition is particularly usefulfor preventing or reducing haemorrhage and/or bleeding associated withthe introduction of a mechanical circulatory support device. For thepurposes of the present invention, the preventative or prophylactictreatment can take action before the introduction of the mechanicalcirculatory support device in the patient or after the introductionthereof, i.e. in the days following the introduction of the device, butin any case before the occurrence of bleeding or haemorrhage.

In a preferred embodiment, the pharmaceutical composition comprising vonWillebrand factor is administered to the patient with mechanicalcirculatory support at a dose of at least 30 IU/kg of body weight.

By “administration” is meant the injection into the patient of thepharmaceutical composition according to the invention. Thisadministration comprises parenteral injections, such as in particularintravenous, intramuscular, sub-cutaneous, intraorbital, intradermal,intra-spinal and intraperitoneal injections as well as infusion directlyinto a tissue or an organ. Administration orally or by using theairways, for example by inhalation, are also envisaged within the scopeof the present invention. Particularly advantageously, thepharmaceutical composition comprising von Willebrand factor isadministered intravenously.

Advantageously, the pharmaceutical composition comprising von Willebrandfactor according to the invention is administered to the patient at adose of at least 40 IU/kg of body weight, preferably 45 IU/kg of bodyweight, preferably 50 IU/kg of body weight, preferably 55 IU/kg of bodyweight, preferably 60 IU/kg of body weight, preferably 65 IU/kg of bodyweight. Particularly advantageously, the dose is 50 IU/kg of bodyweight. A person skilled in the art will know how to adapt the dosedepending on the administration route chosen. In particular, ifintravenous administration is chosen, the dose preferably administeredwould be 50 IU/kg of body weight.

Advantageously, the dosing regime provides a modification of themultimer distribution of von Willebrand factor with an increase of 20%in the multimers larger than 15 mers for 1 hour after the injection.Advantageously, the dosing regime provides a modification of themultimer distribution of von Willebrand factor with an increase in themultimers larger than 15 mers for at least 30 mins. Preferably, thedosing regime provides a modification of the multimer distribution ofvon Willebrand factor with an increase in the multimers larger than 15mers for at least 45 mins. Advantageously, the dosing regime provides amodification of the multimer distribution of von Willebrand factor withan increase in the multimers larger than 15 mers for at least 1 hourafter the injection. This temporary increase and this modification makeit possible to re-establish a normal angiogenesis in themicrocirculation.

Advantageously, the dosing regime makes it possible to restore normalfunctionality of the von Willebrand factor measured by a VWF:Act/VWF:Agresult >0.7. Advantageously, the mean plasma concentration of vonWillebrand factor and the maximum mean plasma concentration of vonWillebrand factor are measured after the intravenous administration ofthe pharmaceutical composition comprising von Willebrand factoraccording to the invention.

In a particularly advantageous embodiment, the first administration ofthe pharmaceutical composition comprising von Willebrand factor iscarried out before the occurrence of the first haemorrhage or the firstbleeding in the patient with the patient's mechanical circulatorysupport.

Advantageously, the first administration of the pharmaceuticalcomposition comprising von Willebrand factor is carried out four daysafter the introduction of the patient's mechanical circulatory support.

Advantageously, the first administration of the pharmaceuticalcomposition comprising von Willebrand factor is carried out five daysafter the introduction of the patient's mechanical circulatory support,preferably six days, preferably seven days after the introduction of thepatient's mechanical circulatory support.

Advantageously, the first administration of the pharmaceuticalcomposition comprising von Willebrand factor is carried out between the4^(th) and the 7^(th) day after the introduction of the patient'smechanical circulatory support. No first administration is carried outbeyond the seventh day. In fact, the administration must be as early aspossible in order to observe an effect, confirming that theeffectiveness is linked to early blocking of angiogenesis. A laterintervention would lead to modulation of the effectiveness.

Advantageously, the first administration of the pharmaceuticalcomposition comprising von Willebrand factor is carried out four toseven days after the introduction of the patient's mechanicalcirculatory support.

Particularly advantageously, the administration of the pharmaceuticalcomposition comprising von Willebrand factor to the patient withmechanical circulatory support is carried out repeatedly at a rate oftwo administrations per week.

Advantageously, if the first administration of the von Willebrand factorcomposition has been carried out on the 4^(th) day after theintroduction of the patient's mechanical circulatory support, then thesecond administration of the von Willebrand factor composition iscarried out between the 5^(th) and the 8^(th) day after the introductionof the patient's mechanical circulatory support.

Advantageously, if the first administration of the von Willebrand factorcomposition has been carried out on the 5^(th) day after theintroduction of the patient's mechanical circulatory support, then thesecond administration of the von Willebrand factor composition iscarried out between the 6^(th) and the 8^(th) day after the introductionof the patient's mechanical circulatory support.

Advantageously, if the first administration of the von Willebrand factorcomposition has been carried out on the 6^(th) day after theintroduction of the patient's mechanical circulatory support, then thesecond administration of the von Willebrand factor composition iscarried out between the 7^(th) and the 8^(th) day after the introductionof the patient's mechanical circulatory support.

Advantageously, if the first administration of the von Willebrand factorcomposition has been carried out on the 7^(th) day after theintroduction of the patient's mechanical circulatory support, then thesecond administration of the von Willebrand factor composition iscarried out on the 8^(th) day after the introduction of the patient'smechanical circulatory support.

Particularly advantageously, the interval between two administrations ofthe pharmaceutical composition comprising von Willebrand factoraccording to the invention must not be less than one day, preferably 2days. Particularly advantageously, the interval between twoadministrations of the pharmaceutical composition comprising vonWillebrand factor according to the invention must not be greater thansix days, preferably five days, preferably four days, preferably threedays.

Particularly advantageously, the administration of the pharmaceuticalcomposition comprising von Willebrand factor to the patient withmechanical circulatory support is carried out repeatedly at a rate oftwo administrations per week for at least 15 days.

Advantageously, the administration of the pharmaceutical compositioncomprising von Willebrand factor to the patient with mechanicalcirculatory support is carried out repeatedly at a rate of twoadministrations per week for at least 30 days, preferably 45 days,preferably 60 days, preferably 75 days, preferably 90 days.

In a particularly advantageous embodiment, the pharmaceuticalcomposition comprising von Willebrand factor according to the inventionis administered to the patient with mechanical circulatory supportrepeatedly at a dose of 50 IU/kg of body weight at a rate of twoadministrations per week for 90 days.

The administration of a von Willebrand factor composition in apreventative manner makes it possible to considerably reduce the risksof the occurrence or the recurrence of a haemorrhage and/or bleeding inthese patients. The risks of haemorrhage associated with theintroduction of the mechanical circulatory support are preferablyreduced by 60%. This preventative administration of von Willebrandfactor preferably makes it possible to reduce by more than 70%,preferably 80%, preferably 90% the occurrence of haemorrhage and/orbleeding in these patients. Advantageously, this administration of vonWillebrand factor makes it possible to permanently remove any risk ofhaemorrhage and/or bleeding in the patients with mechanical circulatorysupport, including the risks of relapse.

Another subject of the invention relates to a method for thepreventative treatment of haemorrhage and/or bleeding in patients withmechanical circulatory support, comprising the administration to saidpatients of a von Willebrand factor composition.

In a particularly advantageous embodiment, the present invention relatesto the use of a pharmaceutical composition comprising von Willebrandfactor in the manufacture of a medicinal product intended for theprevention of haemorrhage and/or bleeding in patients with mechanicalcirculatory support.

Another aspect of the invention relates to a pharmaceutical compositioncomprising von Willebrand factor intended to be used in the treatment ofhaemorrhage and/or bleeding in patients with mechanical circulatorysupport.

The inventors have found, surprisingly, that the administration of adose of at least 30 IU/kg of body weight at the rate of twoadministrations per week of a pharmaceutical composition comprising vonWillebrand factor to patients with mechanical circulatory supportenabled the effective treatment of haemorrhage and/or bleedingassociated with the introduction of mechanical circulatory support inthese patients.

The present invention also relates to a pharmaceutical compositioncomprising von Willebrand factor intended to be used for the treatmentof haemorrhage and/or bleeding in patients with mechanical circulatorysupport, which comprises the administration to said patients of vonWillebrand factor at a dose of at least 30 IU/kg of body weight at therate of two administrations per week.

The term “treatment” or “curative treatment” is defined as a treatmentleading to a cure or a treatment which alleviates, improves and/oreliminates, reduces and/or stabilizes the symptoms of a disease or thesuffering that it causes.

Advantageously, the pharmaceutical composition comprising von Willebrandfactor according to the invention is administered to the patient at adose of at least 30 IU/kg of body weight, of at least 40 IU/kg of bodyweight, preferably 45 IU/kg of body weight, preferably 50 IU/kg of bodyweight, preferably 55 IU/kg of body weight, preferably 60 IU/kg of bodyweight, preferably 65 IU/kg of body weight. The dose is preferably 50IU/kg of body weight.

Advantageously, the dosing regime provides a modification of themultimer distribution of von Willebrand factor with an increase of atleast 10%, preferably of at least 15%, advantageously of at least 20% ofthe multimers larger than 15 mers lasting for 1 hour after theinjection. Advantageously, the dosing regime provides a modification ofthe multimer distribution of von Willebrand factor with an increase ofthe multimers larger than 15 mers lasting for at least 30 mins.Preferably, the dosing regime provides a modification of the multimerdistribution of von Willebrand factor with an increase of the multimerslarger than 15 mers lasting for at least 45 mins.

Advantageously, the dosing regime provides a modification of themultimer distribution of von Willebrand factor with an increase of themultimers larger than 15 mers lasting for at least 1 hour after theinjection. Advantageously, the dosing regime makes it possible torestore normal functionality of the von Willebrand factor measured by aVWF:Act/VWF:Ag result >0.7.

Advantageously, the mean plasma concentration of von Willebrand factorand the maximum mean plasma concentration of von Willebrand factor aremeasured after the intravenous administration of the pharmaceuticalcomposition comprising von Willebrand factor according to the invention.

Advantageously, the administration of the pharmaceutical compositioncomprising von Willebrand factor to the patient with mechanicalcirculatory support is carried out repeatedly at the rate of twoadministrations per week for at least 15 days.

Advantageously, the administration of the pharmaceutical compositioncomprising von Willebrand factor to the patient with mechanicalcirculatory support is carried out repeatedly at the rate of twoadministrations per week for at least 30 days, preferably 45 days,preferably 60 days, preferably 75 days, preferably 90 days.

In a particularly advantageous embodiment, the pharmaceuticalcomposition comprising von Willebrand factor according to the inventionis administered to the patient with mechanical circulatory support in acurative manner at a dose of 30 IU/kg of body weight repeatedly at therate of two administrations per week for 90 days.

In an advantageous embodiment, the pharmaceutical composition comprisingvon Willebrand factor intended to be used in the treatment ofhaemorrhage and/or bleeding in patients with mechanical circulatorysupport is depleted of factor VIII. Advantageously, the residual amountof factor VIII in the pharmaceutical composition comprising vonWillebrand factor according to the invention is less than or equal to 10IU/100 IU VWF:RCo.

In an advantageous embodiment, the pharmaceutical composition comprisingvon Willebrand factor intended to be used in the treatment ofhaemorrhage and/or bleeding in patients with mechanical circulatorysupport is depleted of ADAMTS13. Advantageously, the residual amount ofADAMTS13 in the pharmaceutical composition comprising von Willebrandfactor is less than or equal to 0.10 ADAMTS13:Act [U/ml].

Another subject of the invention relates to a method for the curativetreatment of haemorrhage and/or bleeding in patients with mechanicalcirculatory support, comprising the administration to said patients of avon Willebrand factor composition.

Advantageously, the method for the curative treatment of haemorrhageand/or bleeding in patients with mechanical circulatory supportcomprises the administration to said patients of a von Willebrand factorcomposition, depleted of blood clotting factor VIII.

Advantageously, the method for the curative treatment of haemorrhageand/or bleeding in patients with mechanical circulatory supportcomprises the administration to said patient of a von Willebrand factorcomposition at a dose of at least 30 IU/kg of body weight, preferably 40IU/kg of body weight, preferably 45 IU/kg of body weight, preferably 50IU/kg of body weight, preferably 55 IU/kg of body weight, preferably 60IU/kg of body weight, preferably 65 IU/kg of body weight. Particularlyadvantageously, the dose is 50 IU/kg of body weight.

Advantageously, the method for the curative treatment of haemorrhageand/or bleeding in patients with mechanical circulatory supportcomprises the administration to said patients of a von Willebrand factorcomposition repeatedly at the rate of two administrations per week.

Advantageously, the method for the curative treatment of haemorrhageand/or bleeding in patients with mechanical circulatory supportcomprises the administration to said patient of a von Willebrand factorcomposition, the first administration of the pharmaceutical compositioncomprising von Willebrand factor being carried out from the occurrenceof the first haemorrhage or the first bleeding in the patient with thepatient's mechanical circulatory support.

In a particularly advantageous embodiment, the method for the curativetreatment comprising the administration to the patient with mechanicalcirculatory support of the pharmaceutical composition comprising vonWillebrand factor according to the invention at a dose of 50 IU/kg ofbody weight repeatedly at the rate of two administrations per week for90 days.

In a particularly advantageous embodiment, the present invention relatesto the use of a pharmaceutical composition comprising von Willebrandfactor intended to be used in the manufacture of a medicinal productintended for the treatment of haemorrhage and/or bleeding in patientswith mechanical circulatory support.

Another aspect, the present invention relates to a process for obtainingthe pharmaceutical composition comprising von Willebrand factor. The vonWillebrand factor composition can be obtained by processes well known toa person skilled in the art, for example by fractionation of plasma, byexpression in cell culture or by expression in the milk of transgenicanimals.

According to a particular embodiment, the von Willebrand factor of theinvention can be obtained from human blood plasma, either from afraction of cryoprecipitated human plasma or from the supernatant ofcryoprecipitated plasma again containing von Willebrand factor orobtained by conventional fractionation methods (Cohn et al., J. Am.Chem. Soc., 68, 459, 1946 and Kistler et al., Vox Sang., 7, 1962,414-424), possibly having been subjected to a pre-purification treatmentin particular by adsorption on aluminium hydroxide, in which the vWF iscomplexed to factor VIII. This is then called “plasmatic” von Willebrandfactor.

In a particular embodiment, the von Willebrand factor is obtained from afraction of cryoprecipitated plasma that has been subjected to a priorpurification step by chromatography using an anion exchanger of theDEAE-Fractogel®-TSK 650 type, as described in the patents EP 0 359 593and EP 0 503 991.

According to another particular embodiment, the von Willebrand factorcan be obtained by genetic engineering, in particular produced by cellsthe DNA of which has been modified by genetic recombination in such away as to express a molecule of FVII, and having the particularcharacteristics of glycosylation. This is then called recombinant vonWillebrand factor. Thus, the von Willebrand factor of the invention isderived from the transcription then the translation of a molecule of DNAcoding for the von Willebrand factor in a host cell. The recombinant vonWillebrand factor of the invention can be obtained using standardtechniques well known to a person skilled in the art, allowing theexpression of a protein in a biological system. More particularly, by“recombinant von Willebrand factor” is meant any von Willebrand factorobtained by genetic recombination and expressed by a cultured cell line.The following lines can be mentioned by way of example: BHK (BabyHamster Kidney) and in particular BHK tk″tsl3 (CRL 10314, Waechter andBaserga, Proc. Natl. Acad. Sci. USA 79:1106-1110, 1982), CHO (ATCC CCL61), COS-I (ATCC CRL 1650), HEK293 (ATCC CRL 1573; Graham et al, J. Gen.Virol. 36:59-72, 1977), Rat Hep I (Rat hepatoma; ATCC CRL 1600), Rat HepII (Rat hepatoma; ATCC CRL 1548), TCMK (ATCC CCL 139), Human lung (ATCCHB 8065), NCTC 1469 (ATCC CCL 9.1) and DUKX cells (CHO cell line)(Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216-4220, 1980), 3T3cells, Namalwa cells, or BHK cells that have been adapted to grow in theabsence of serum (document U.S. Pat. No. 6,903,069). Recombinant vonWillebrand factor can thus be isolated from a fraction enriched with vWFand/or with factor VIII/recombinant vWF complex isolated from thesupernatant of cell cultures according to known techniques.

In another particular embodiment of the invention, the von Willebrandfactor is a transgenic von Willebrand factor, i.e. obtained by geneticrecombination and expressed by a living tissue, either animal or plant.

Advantageously, the transgenic von Willebrand factor is obtained bygenetic recombination and expressed in an animal, called a transgenicanimal. By transgenic animal is meant an animal into the genome of whichone or more genes has been introduced by transgenesis. Preferably, thevon Willebrand factor is in particular produced in the milk of atransgenic animal, the formulation of the invention making it possibleto retain a satisfactory level of biological activity of the vonWillebrand factor after the lyophilization thereof. According to apreferred embodiment, the human von Willebrand factor is produced in themilk of non-human female transgenic mammals, genetically modified inorder to produce this protein. The rabbit, goat, cow, rodent, hamster,camel, llama, Dromedary camel, mouse, rat, pig, sow, horse, dog, cat,ewe, ruminants, hog etc., the list not being limitative, can inparticular be mentioned as examples of genetically modified non-humantransgenic mammals capable of producing this protein. It is preferablythe is milk of a transgenic rabbit or goat. The secretion of vonWillebrand factor by the mammary glands, making possible the secretionthereof in the milk of the transgenic mammal, implies that theexpression of the von Willebrand factor can be controlled in atissue-dependent manner. Such control methods are well known to a personskilled in the art. The expression is controlled by the sequencesallowing the expression of the protein in a particular tissue of theanimal. These are, in particular, the WAP promoter sequences, betacasein sequences, beta lactoglobulin sequences and signal peptidesequences. The process for extracting the proteins of interest from themilk of the transgenic animals is described in the patent EP 0 264 166.Transgenic von Willebrand factor can also be isolated from a fractionenriched with vWF and/or with factor VIII/vWF complex obtained from themilk of transgenic mammals as described in the U.S. Pat. No. 6,518,482.

Whatever the method of obtaining the von Willebrand factor, from plasmaor by genetic engineering (recombinant or transgenic), the vonWillebrand factor fraction can be diafiltered in order to incorporatethe appropriate excipients intended to allow the von Willebrand factorto be heated to dryness without the risk of denaturation, concentratedby ultrafiltration, packaged in flasks and lyophilized, after a prioraddition of an additional pharmaceutically acceptable stabilizer, suchas albumin.

Finally, the lyophilizates undergo a final step of viral inactivation byheating the lyophilizate to dryness according to standard conditions, at80° C. for 72 hours, in order to inactivate the non-enveloped viruseswhich have not been inactivated and/or eliminated by at least one of thetwo preceding steps for the inactivation and/or elimination of viruses.The lyophilizates heated to dryness are then reconstituted in an aqueousmedium compatible with clinical use, preferably in 10 ml of water forinjection (WFI) in order to obtain an injectable formulation.

The injectable formulation can be administered parenterally, preferablyby intravenous, sub-cutaneous or intramuscular route. The administrationof the liquid form (solution or suspension, before drying) or the powderform, by any appropriate route or means, is not excluded. In aparticular embodiment of the invention, the injectable formulation isadministered intravenously.

EXAMPLES Example 1: Preparation of a von Willebrand Factor Composition

1) Obtaining a Fraction Containing vWF

A cryoprecipitate of human plasma is used, resuspended in an aqueousheparin sodium solution (2 U/ml), at a pH of 7-7.1.

This cryoprecipitate solution is subjected to a pre-purification onaluminium hydroxide in order to eliminate the main contaminants, asdescribed in the patent EP 0 359 593. The pre-purified supernatant isthen recovered and is subjected to a standard viral inactivationtreatment by solvent-detergent, in the presence of Tween®-TNBP.

The pre-purified cryoprecipitate solution is then injected into aFractogel® TSK-DEAE 650 (M) type chromatography column with a length of25 cm and a diameter of 1 cm, equilibrated beforehand with a bufferconstituted by 0.01 M trisodium citrate, 0.001 M calcium chloride, 0.11M sodium chloride, 0.12 M glycine and 0.016 M lysine, adjusted to a pHof 7.01, the linear velocity of the mobile phase of which is set at 100cm/hour. The von Willebrand factor, the factor VIII and the fibronectinare retained on the chromatographic support. The weakly-retainedproteins or those not retained by the support, principally thefibrinogen and the immunoglobulin Gs (IgGs), are eliminated in thefiltrate, as well as the Tween® and TNBP, by several successive washingswith the same buffer.

The protein content is monitored by measuring the absorption at 280 nm(labelled O.D. below).

When the O.D., measured at 280 nm, has returned to the baseline, theconcentration of sodium chloride in the buffer is increased to 0.15 M.Under these conditions, the von Willebrand factor is eluted. The eluatethus obtained is very rich in von Willebrand factor and in fibronectinand also contains Tween® and TNBP, as well as residual factor VIII.

2) Chromatographic Separation

The previously-eluted fraction enriched with von Willebrand factor,constituting one batch of starting fraction containing the vonWillebrand factor according to the invention, is loaded into aDEAE-Fractogel®-TSK 650 (M) type chromatography column with a length of25 cm and a diameter of 1 cm, equilibrated beforehand with the samebuffer as that in 1), the osmolarity of which is 387 mosmolkg⁻¹, thelinear velocity of which is set at 100 cm/hour. 140 ml of this fractioncontaining 12.9 IU vWF/ml and 6.6 IU factor VII/ml, or an R ratio of51.1% (R=FVIII:C/FvW:RCo) is injected.

The column is then washed with an acidic buffer of 20 mM sodium acetate,adjusted to a pH of 4.35 and 80 mosmolkg⁻¹, with a linear velocity of150 cm/hour. Under these conditions, a very good elimination not only ofthe viral inactivation agent residues but also of the fibronectin andabove all of factor VIII which was again complexed to the von Willebrandfactor is ensured, without precipitation of these proteins beingobserved in the column. When the O.D. has returned to the baseline, thelinear velocity is brought back to 100 cm/hour then the column is rinsedand equilibrated with the same buffer as previously, containing NaCl at0.11 M.

The fraction containing the von Willebrand factor is eluted byincreasing the concentration of the NaCl of the equilibrating buffer to0.17 M, adjusting to a pH of 6.95 and 492 mosmolkg⁻¹.

The eluted von Willebrand factor fraction then undergoes standardsterilizing filtration treatments on filters of 0.22 μm, nanofiltrationon filters of 35 nm, diafiltration and ultrafiltration according toknown techniques so that the von Willebrand factor concentrate has aspecific activity (S.A.) of at least 90 IU RCo/mg of protein.

Albumin at 10 g/l is added to the von Willebrand factor concentratesthus obtained then it is lyophilized at −40° C. for 48 hours. Thelyophilization is followed by a heat viral inactivation treatment byheating the lyophilizate to dryness at 80° C. for 72 hours.

Example 2: Effect of the Administration of a Von Willebrand FactorComposition on the Prevention of Haemorrhage and/or Bleeding in Patientswith Mechanical Circulatory Support

The present study is based on 136 patients over the age of 18 andrequiring the introduction of mechanical circulatory support due toadvanced heart failure.

In the following, the abbreviation d means “day”. The abbreviation d=0corresponds to the day of surgery, thus-named day 0. By way of example,the abbreviation d+4 corresponds to the 4^(th) day after surgery.

On d=0 the patients have the mechanical circulatory support deviceimplanted through the implantation of a HeartMate II continuous flowpump. Between d+4 and d+7 the patients received a first administrationof the pharmaceutical composition comprising von Willebrand factoraccording to the invention at a dose of 50 IU/kg of body weight. Theadministration of this pharmaceutical composition comprising vonWillebrand factor was carried out at the rate of two administrations perweek and at a dose of 50 IU/kg of body weight for 90 days.

The occurrence of haemorrhage and/or bleeding was monitored for each ofthe patients for 90 days. In addition, the development of the angiogenicmarkers (VEGF, angiopoietin 2, galectin 1 and 3) as well as thedevelopment of the pharmacological characteristics of the von Willebrandfactor (development of the vWF:Act, vWF:Ag, vWF:CBA ratios and thelevels of high-molecular weight (HMW) multimers were monitored at d+15,d+30, d+45, d+60, d+75 and d+90.

The results show that the administration of 50 IU/kg of the vonWillebrand factor composition according to the invention at the rate oftwo administrations per week for 90 days makes it possible to preventthe occurrence of haemorrhage and/or bleeding in these patients.

1.-17. (canceled)
 18. A pharmaceutical composition comprising vonWillebrand factor in an amount effective for treating or preventinghaemorrhage and/or bleeding in patients with mechanical circulatorysupport, wherein: the composition contains a residual amount of factorVIII less than or equal to 10 IU/100 IU VWF:RCo.
 19. The pharmaceuticalcomposition according to claim 18, wherein the pharmaceuticalcomposition is depleted of factor VIII.
 20. The pharmaceuticalcomposition according to claim 18, wherein the pharmaceuticalcomposition is depleted of ADAMTS
 13. 21. The pharmaceutical compositionaccording to claim 18, wherein high-molecular weight multimers representat least 65% of total multimer content of von Willebrand factorcontained in the pharmaceutical composition.
 22. The pharmaceuticalcomposition according to claim 21, wherein the content of high-molecularweight multimers is close to that of plasma.
 23. A method of treating orpreventing haemorrhage and/or bleeding in patients with mechanicalcirculatory support, comprising administering to the patient thepharmaceutical composition according to claim
 18. 24. The methodaccording to claim 23, wherein the composition is administered to thepatient at a dose of at least 30 IU/kg of body weight.
 25. The methodaccording to claim 24, wherein the dose is at least 40 IU/kg of bodyweight.
 26. The method according to claim 24, wherein the dose is atleast 50 IU/kg of body weight.
 27. The method according to claim 24,wherein the dose is at least 60 IU/kg of body weight.
 28. The methodaccording to claim 24, wherein the dose is administered repeatedly at arate of two administrations per week.
 29. The method according to claim23, wherein the pharmaceutical composition is first administered four toseven days after the mechanical circulatory support is introduced intothe patient.
 30. The method according to claim 23, wherein thecomposition is administered intravenously.
 31. The method according toclaim 23, wherein the composition is depleted of factor VIII.
 32. Themethod according to claim 23, wherein the composition is depleted ofADAMTS
 13. 33. The method according to claim 23, wherein high-molecularweight multimers represent at least 65% of total multimer content of vonWillebrand factor contained in the pharmaceutical composition.
 34. Themethod according to claim 23, wherein high-molecular weight multimersrepresent at least 70% of total multimer content of von Willebrandfactor contained in the pharmaceutical composition.
 35. The methodaccording to claim 23, wherein high-molecular weight multimers representat least 75% of total multimer content of von Willebrand factorcontained in the pharmaceutical composition.
 36. The method according toclaim 23, wherein the content of high-molecular weight multimers isclose to that of plasma.
 37. The method according to claim 23, whereinthe high-molecular weight multimers are present in amounts sufficientfor vivo activity.